these slides can easily give better understanding to the reader about the Steps involved in Southern Blotting.

1. SOUTHERN
BLOTTING
Muhammad Ali
Department of Bioinformatics
Govt Postgraduate College Mandian Abbottabad

2. CONTENTS
• INTRODUCTION
• HISTORY
• PURPOSE OR FUNCTION
• PROTOCOL
• APPLICATIONS

3. INTRODUCTION
• DEFINITION
A Southern blotting is a method used in molecular biology for
detection of a specific DNA sequence in DNA samples. Southern blotting
combines transfer of electrophoresis separated DNA fragments to a filter
membrane and subsequent fragment detection by probe hybridization.
The DNA detected can be a single gene, or it can be part of a larger piece of
DNA such as a viral genome.

4. HISTORY
• This technique was discovered by Edward M Southern in 1973
• His work was published in 1975

5. PURPOSE OR FUNCTION
• To determine the DNA sequence between 2 DNA samples .
• Whether the DNA sequence is present or not.
• Also used for the detection of specific restriction fragments

6. PROTOCOL
• Extract the DNA from the cell
• Digest the DNA with restriction endonuclease
• Mixture of fragment subjected to gel electrophoresis
• Gel separates the fragments according to size
• The restriction fragments present in the gel are denatured with alkaline
solution

7. CONTI…
• Transfer the DNA on the nitrocellulose or nylon paper
• Hybridization of single strand of DNA with probe.
• Visualization

8. DNA EXTRACTION AND
DIGESTION
• First we will take out the complex DNA structure from
the cell
• This process will occur with the help of detergents
• or buffers
• Restriction enzymes are used to cut the DNA into
smaller fragments

9. GEL ELECTROPHORESIS
• Add the fragments in the wells/groves of gel
• Pass the electric current through the gel
• Fragments will b separated according to size

10. IMPRINTING ON NYLON PAPER
• Take out the gel from the tray
• Take another tray filled with buffer
• Place sponge in buffer and put gel on it
• Put the gel under the nylon paper for imprinting
• Put weight on it

11. CONTI…
• After several hours, the DNA fragments will be imprinted on
paper
• The blot will be permanent by
• Drying at ~80°C
• Exposing to UV irradiation

13. HYBRIDIZATION
• Take the imprinted nitro cellulose
• Subject the Radio labelled probe toward the nitro cellulose
• The probe will hybridize with its complimentary fragment
• Wash the paper to remove the excessive probes
• After hybridization, the paper will be ready for visualization.

14. VISUALIZATION
• Expose the filter or nitrocellulose to the
X RAY film
• By this, only the area hybridized with probe
will be highlighted
• We can visualize our targeted protein

15. APPLICATIONS
• Paternity and Maternity Testing
• Criminal Identification and Forensics
• Personal Identification
• Diagnosis of HIV-1 and infectious disease

16. Any
Questions
..???